敲低BRIX1对三阴型乳腺癌细胞增殖和凋亡的影响

Effects of Knockdown BRIX1 on Proliferation and Apoptosis of Triple Negative Breast Cancer Cells

目的通过体外细胞实验观察BRIX1基因对三阴性乳腺癌(triple negative breast cancer,TNBC)细胞MDA-MB-231的增殖与凋亡的影响。方法将MDA-MB-231细胞株分为实验组(shBRIX1组)及对照组(shCtrl组)。shBRIX1组采用构建敲低BRIX1基因表达的慢病毒载体转染MDA-MB-231细胞,以敲低BRIX1表达;shCtrl组采用慢病毒空载体转染MDA-MB-231细胞。应用实时荧光定量PCR(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)及Western Blot方法检测两组的转染效率;采用Celigo细胞成像技术、噻唑蓝(methyl thiazolyl tetrazolium,MTT)实验连续5 d检测两组细胞的增殖情况;采用细胞克隆实验检测细胞克隆形成数;采用流式细胞仪荧光分选(fluorescence activated cell sorting,FACS)技术和Caspase3/7活性检测比较两组细胞的凋亡能力。结果与shCtrl组细胞相比,敲低BRIX1表达的慢病毒载体转染MDA-MB-231细胞72 h后,RT-qPCR检测shBRIX1组中细胞的BRIX1基因表达水平明显下降(P<0.05);Western Blot检测shBRIX1组细胞中的BRIX1蛋白表达水平下降。敲低BRIX1后,Celigo、MTT及细胞克隆实验结果显示从第二天开始MDA-MB-231细胞增殖能力较shCtrl组明显下降(P<0.01);FACS实验及Caspase3/7活性检测结果显示敲低BRIX1组的MDA-MB-231凋亡细胞数量增加(P<0.05)。结论下调BRIX1能抑制三阴型乳腺癌细胞增殖,促进凋亡,为寻找TNBC治疗新靶点提供实验依据。

Objective To investigate the effects of BRIX1 on the proliferation and apoptosis of triple negative breast cancer(TNBC)cell line MDA-MB-231 in vitro.Methods MDA-MB-231 cells were divided into the experimental group(shBRIX1 group)and the control group(shCtrl group).In the shBRIX1 group,MDA-MB-231 cells were transfected with lentivirus vector constructed by knocking down BRIX1 gene expression;in the shCtrl group,MDA-MB-231 cells were transfected with lentivirus empty vector.The transfection efficiency was detected by real time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot.The cell proliferation of the two groups were tested for five days by the Celligo assay and the MTT assay.The number of cell clone formation was detected by cell cloning experiment.And the apoptotic ability of cells was detected by FACS and Caspase 3/7 activity.Results 72 hours after transfection of MDA-MB-231 cells with knockdown BRIX1 lentivirus expression vector,the expression of BRIX1 gene in the shBRIX1 group decreased significantly compared with the shCtrl group(P<0.05).The protein BRIX1 expression level in shBRIX1-group cells was lower than that of the shCtrl group which were detected by Western blot(P<0.01).After knocking down BRIX1,the results of Celigo,MTT and cell cloning showed that the proliferation ability of MDA-MB-231 cells was significantly lower than that of the shCtrl group from the second day(P<0.01);FACS test and Caspase 3/7 activity test showed that the number of apoptotic cells in the BRIX1-knockdown group increased significantly compared with the shCtrl group(P<0.05).Conclusion Down regulation of BRIX1 could inhibit the proliferation and promote apoptosis of TNBC cells,which provides experimental data for new targets of TNBC treatment.